The introduction of luminescent semiconductor nanocrystals or quantum dots (QDs) to biology has provided researchers with novel fluorescent tools for potentially achieving advances in imaging and sensing. See, for example, U.S. Patent Application Publication Nos. 2008/0087843 and 2011/0089241, each of which is incorporated herein by reference. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors.
FRET has been used in a wide variety of applications, including: static distance measurements within or between (bio)molecules (i.e. FRET as a “spectroscopic ruler” between ˜1-10 nm); dynamic observation of changes in biomolecular conformation; diagnostic constructs that use changes between FRET “on” (high efficiency) and “off” (low efficiency) states to detect chemical/biological analytes; and light harvesting/photonic wires. In spectroscopic ruler contexts, FRET is generally implemented in its simplest configuration, which comprises a single donor luminophore and single acceptor chromophore. The FRET efficiency can be used to derive the donor-acceptor separation distance and is almost always measured on the basis of quenching of the donor luminescence intensity or decrease in the donor excited state lifetime. When the acceptor chromophore is also fluorescent, the ratio of acceptor and donor luminescence intensities can be a useful qualitative or quantitative measure. Diagnostic probes (for example, molecular beacons, Scorpion primers, or TaqMan probes) also predominately utilize discrete donor-acceptor pairs. Multi-step FRET relays have been described previously, using only prompt (nanosecond scale) fluorescence. The primary purpose has been to extend the net range of FRET or serve as a photonic wire.
Described herein is a time-gated, two-step FRET relay effective to provide temporal transference of a prompt FRET pathway, or provide spectro-temporal encoding.